Although it is widely accepted that B cells with self-reactivity are deleted or rendered functionally inactive, self-reactive antibodies, referred to as "natural autoantibodies" can be found in the serum of healthy animals. Such natural autoantibodies are produced by a small fraction of B cells with distinctive expression of the cell surface glycoprotein CD5. These CD5+ autoreactive B cells are more frequently generated from fetal B cell precursors as a part of "B-1" B cell development than those in the adult bone marrow. The importance of fetal/neonatal natural autoantibodies in protective immunity is exemplified by T15 idiotype positive anti-phosphorylcholine antibody, the most protective antibody to virulent pnuemococcal infection, rapidly produced after infection. Our research explores why such autoreactive B cells exist naturally, the mechanism whereby they develop, and their potential for dysregulation. By establishing and investigating mouse natural autoreactive B cell models expressing natural anti-Thy-1 autoantibody (ATA), we found that self-antigen is important for CD5+ ATA autoreactive B-1 B cell accumulation with relatively higher B cell receptor (BCR) signal intensity mediating a positive selection process. In contrast, negative selection occurs for cells expressing the identical BCR during conventional B cell ("B-2") development in spleen from bone marrow precursors. In this renewal, we will obtain a comprehensive understanding of the mechanism for development of this B cell subset . We will first investigate the mechanism of follicular B cell maturation and maintenance in B-2 B cell development. The significance of non-BCR signaling, provided by other lymphocytes, T cells and B-1 B cells, for the maturation of B cells that lack a BCR crosslinking signal, and a role for bacterial products in B cell survival will be examined (Aim 1). To understand why autoreactive B-1 positive selection occurs from fetal B cell development, we will identify a "fetal B-1 signature" that we hypothesize reflects distinctive cellular machinery determining a difference in the BCR signaling threshold, relative to bone marrow B-2 development. The fate of such earlygenerated B-1 cells will be examined in Lysmd2-GFP reporter mice, based on our recent identification of this gene as a component of the fetal B-1 signature (Aim 2). Accomplishing these aims will significantly advance our understanding of B cell development, and the importance of self-antigen and microenvironment in establishing a fully competent immune system. In humans, CD5 expression is a hallmark of late developing chronic B cell leukemia ( B CLL). Arriving at a comprehensive understanding of B cell development and the mechanism of their maintenance will be critically important for designing rational therapies of such dysregulated CD5+ B cells in the future. PUBLIC HEALTH RELLEVANCE: The immune system plays a crucial role in the acute response to infectious agents and natural autoreactive B-1 B cells play a key role in this system, rapidly producing antibodies to eradicate microorganisms. However, abnormal expansions of B-1 B cells can occur with age in certain autoimmune mouse strains, sometimes progressing to CD5+ B leukemia/lymphoma. Arriving at a comprehensive understanding of the mechanism(s) of B cell development, and establishing how natural autoreactive B-1 B cell are normally regulated and function, as proposed in this application, will be critically important both for designing rational therapies of infectious disease and for treating dysregulated B cell expansions that may lead to cancer.